The purpose of this investigation is to determine biochemical mechanisms of contractile responses to hormones and drugs in vascular and airway smooth muscles which have their actions mediated by Ca2 ion and cyclic AMP. The primary focus will be directed toward elucidation of the physiological roles of protein phosphorylation. Ca2 ion is required for myosin light chain kinase and phosphorylase kinase activities. Furthermore, phosphorylation of smooth muscle myosin is obligatory for actin activation of myosin Mg2 ion-dependent ATPase activity which implies myosin phosphorylation may be essential for contraction. Myosin light chain kinase and phosphorylase kinase are both phosphorylated by cyclin AMP-dependent protein kinase which leads to inhibition or activation, respectively, of the two enzymes. Dose and temporal responses of smooth muscle contraction will be obtained with agents which activate different pharmacological receptor systems or with ionic perturbations. After quick-freezing isolated smooth muscles cyclic AMP content, activation of cyclic AMP-dependent protein kinase and phosphorylation of myosin light chain, phosphorylase kinase and myosin light chain kinase will be measured. Biochemical studies (physical and enzymatic properties) will also be performed on purified myosin light chain kinase, myosin light chain phosphatase and phosphorylase kinase from vascular and airway smooth muscles. We will determine the basis for the isozymic differentiation of the same enzmes found in skeletal and cardiac muscles. The mechanisms of activation of the two kinases by Ca2 ion and CDR (Ca2 ion-dependent regulatory protein) will be compared as well as regulation of the enzymatic activities via phosphorylation by cyclic AMP-dependent protein kinase. These results will provide a framework by which biochemical mechanisms of regulating myosin light chain kinase and phosphorylase kinase by Ca2 ion and cyclic AMP may be related to contractile state in living smooth muscle. In addition, these studies will determine if myosin phosphorylation itself could be a primary event in contractile responses to diverse pharmacological agents.